Detection of Global DNA Methylation and Paternally Imprinted H19 Gene Methylation in Preeclamptic Placentas

ORIGINAL ARTICLE

Detection of global DNA methylation and paternally imprinted H19 gene methylation in preeclamptic placentas

Wen-long Gao1,4, Dong Li1,4, Zhong-xin Xiao2, Qin-ping Liao3, Hui-xia Yang3, Yu-xia Li1, Lei Ji1 and Yan-ling Wang1

Preeclampsia (PE) is a severe hypertensive disorder associated with pregnancy; despite substantial research effort in the past several years, the etiology of PE is still unclear. The role of epigenetic factors in the etiology of PE, including DNA methylation, has been poorly characterized. In the present study, we investigated global DNA methylation as well as DNA methylation of the paternally imprinted H19 gene in preeclamptic placentas. Using 5-methylcytosine immunohistochemistry and Alu and LINE-1 repeat pyrosequencing, we found that the global DNA methylation level and the DNA (cytosine-5) methyltransferase 1 mRNA level were signiﬁcantly higher in the early-onset preeclamptic placentas when compared with the normal controls. Data from methylation-sensitive high resolution melting demonstrated hypermethylation of the promoter region of the H19 gene, and results of real-time PCR showed decreased mRNA expression of H19 gene in the early-onset preeclamptic placentas as compared with the normal controls. Our results suggest that abnormal DNA methylation during placentation might be involved in the pathophysiology of PE, especially early-onset preeclampsia. Hypertension Research (2011) 34, 655–661; doi:10.1038/hr.2011.9; published online 17 February 2011

Keywords: DNA methylation; H19; placenta; preeclampsia

INTRODUCTION logical and biochemical changes of trophoblast cells and endometrial Preeclampsia (PE) is a pregnancy-specific syndrome, generally defined cells, thus negatively impinging upon the delicate feto-maternal as the development of hypertension and proteinuria after 20 weeks interaction during the early pregnancy. In addition, results from of gestation in a previously normotensive woman. The disease affects cloned animals also revealed that disruptions in DNA methylation 5–7% of pregnancies worldwide, and is one of the major causes of the profiles in placentas may be involved in the severe symptoms of maternal and neonatal mortality and morbidity.1,2 PE has been termed placentomegaly.8,9 the ‘disease of theories’, reflecting the confusion that surrounds the Recent mounting evidence strongly suggests the contribution of etiology of PE.3 The pathogenesis of PE is complex, likely resulting epigenetic factors to the etiology of PE. Results from two independent from the interaction of numerous genetic, immunologic and environ- laboratories demonstrated that significant hypomethylation occurs mental factors.4 Although the prime cause remains unclear, it is in the promoter regions of the serpinb5 and serpina3 genes in generally accepted that the syndrome may be initiated by abnormal preeclamptic placenta as compared with normal controls placen- placental development during the first trimester, resulting in placental tas.10,11 Novakovic et al. reported decreased DNA methylation levels insufficiency and the release of impaired placental factors into the of the cyp24a1 gene in preeclamptic placenta tissues.12 However, most maternal circulation.5–7 of these evidences are limited to the analysis of individual genes, and It has been well documented that a successful pregnancy involves a variations of global DNA methylation is still poorly understood in PE. complex and precisely coordinated modulation of gene expression in On the other hand, parent-of-origin specific imprinting of many gestational tissues mainly including placenta and uterine decidua. genes in placenta is likely to have key roles in many aspects of placental Recent evidence suggests that epigenetic regulation of gene expression function, including trophoblast cell proliferation, differentiation, is likely to have a key role in this process. Serman et al. indicated that angiogenesis and transportation of nutrients.13,14 The variations of variations in the extent of DNA methylation could induce morpho- some imprinted genes were also reported to be associated with PE.

1State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 2School of Public Health and Family Medicine, Capital Medical University, Beijing, China; 3Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing, China and 4Graduate School of Chinese Academy of Sciences, Beijing, China Correspondence: Dr L Ji or Dr Y-l Wang, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Beijing 100101, China. E-mails: [email protected] or [email protected] Received 5 August 2010; revised 13 December 2010; accepted 16 December 2010; published online 17 February 2011 Detection of DNA methylation in preeclampsia W-l Gao et al 656

These mainly included the single nucleotide polymorphorism of the peroxide for 15 min, incubated with 1% bovine serum albumin for 1 h at room STOX1 gene (van Dijk et al.)15 and the gene mutation of the parentally temperature, and subsequently incubated with a mouse monoclonal antibody imprinted p57 gene (Kanayama et al.)16 The paternally imprinted H19 against 5-MC (0.5 mgmlÀ1)at41C overnight. The negative control was gene is transcribed as an untranslated RNA that may serve as a prepared by replacing primary antibodies with non-immune mouse IgG at riboregulator. The gene is located in close proximity to the maternally the same concentration. The slides were further incubated with horseradish peroxidase streptavidin -conjugated anti-mouse IgG (Zhongshan Goldenbrige imprinted IGF-2 gene on chromosome 11p15.5.17 Notably, the H19 18 Biotechnology, Beijing, China) for 30 min. Final visualization was performed by null mice display hyperplasia of all layers of the placenta, and recent incubating the sections with a DAB Detection Kit (Zhongshan Goldenbrige studies demonstrated the regulatory role of H19 in the invasive Biotechnology), and the staining time was tightly ﬁxed at 5 min for every slide. 19,20 property of placental trophoblasts. The evidences strongly indicate The slides were evaluated under a light microscope (Olympus BX51, Tokyo, that the H19 gene is critical to the early development of placenta. Japan), and the staining intensity of the antibody was analyzed by a semi- However, it remains unclear as to the DNA methylation status in the quantitative analysis HSCORE.22 The HSCORE was calculated using the promoter region of H19 gene in preeclamptic placenta. following equation: HSCORE¼SPi (i+1), where i was the staining intensity In the present study, we investigated the extent of global DNA (1¼weak, 2¼moderate and 3¼strong), and Pi was the percentage of the methylation in preeclamptic placentas obtained from a cohort of positively stained cells for each intensity varying from 0 to 100%. The Chinese patients, and further compared the mRNA expression as assessment of staining intensity and HSCORE were evaluated in a double- blind condition by three independent observers who did not know the disease well as the methylation change in the promoter region of the paternally status of the samples. imprinted H19 gene between preeclamptic and normal placentas.

RNA isolation and real-time PCR METHODS Total RNAs from cells and placenta tissue were isolated with TRIzol reagent Subjects (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Placentas from normal and severe preeclamptic pregnant women were obtained A total of 2 mm of total RNA was used for reverse transcription with oligo- from the Department of Obstetrics and Gynecology, Peking University First (dT)15 primer using moloney murine leukemia virus reverse transcriptase Hospital, China, from November 2005 to March 2007. The study was approved (Promega, Madison, WI, USA). Real-time quantitative PCR was performed by the Research Ethic Committees of the Institute of Zoology, Chinese using the SYBR green detection system (TaKaRa Biotechnology, Dalian, China) Academy of Sciences and Peking University First Hospital. A total of 24 in the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster preeclamptic cases and 24 controls were recruited for a nested case-control City, CA, USA). Reaction for each sample was performed in duplicate in a 20 ml study. According to the onset time of the clinical symptoms, the 24 pre- reaction volume containing 1 ml cDNA, 10 ml SYBR Premix Ex Taq, 0.4 mlROX eclamptic cases were categorized into 10 early-onset (E-PE, 34 weeks) and p Reference Dye II (50Â) and 0.2 mM of each primer. The PCR conditions and 14 late-onset PE (L-PE, 434 weeks). Severe PE was diagnosed according to primers sequence are shown in Table 2. To control for uniform amount of the criterion of the International Society for the Study of Hypertension in Pregnancy. In brief, these study patients had no history of preexisting or chronic hypertension, but showed systolic blood pressure of 4160 mm Hg Table 2 Primers and PCR conditions for real-time PCR or diastolic blood pressure of 4110 mm Hg on at least two occasions, PCR accompanied by significant proteinuria (42 g per 24 h or 3+ by dipstick in two random samples collected at 44 h interval) after 20 weeks of gestation. Gene PCR primers (5 ¢–3 ¢) PCR conditions production Those who developed renal disease, transient hypertension in pregnancy, DNMT1 F: TACCTGGACGACCCTGACCTC Real-time PCR: 103 bp gestational diabetes, spontaneous abortion, intrauterine fetal death, fetal R: CGTTGGCATCAAAGATGGACA 95 1C for 5 s, chromosomal or congenital abnormalities or pregnancies conceived by fertility DNMT3a F: GACAAGAATGCCACCAAAGC60 1C for 30 s. 190 bp treatment were excluded from this study. The clinical characteristics of the patients included in this study were summarized in Table 1. Placental R: CGTCTCCGAACCACATGAC (40 cycles) Specimens were quickly dissected, snap frozen in liquid nitrogen and stored at DNMT3b F: GACTCGAAGACGCACAGCTG 98 bp À80 1C until prepared for further analyses. R: CTCGGTCTTTGCCGTTGTTATAG H19 F: CCGGACACAAAACCCTCTAGCT 142 bp R:TGTTCCGATGGTGTCTTTGATG Immunohistochemistry for 5-methylcytosine (5-MC) GAPDH F: CCACATCGCTCAGACACCAT 114 bp Immunohistochemistry was performed as previously described.21 Briefly, 4 mm R: GGCAACAATATCCACTTTACCAGAGT thick tissue frozen sections were mounted onto microscope glass slides and b-actin F: CCTGGCACCCAGCACAAT 70 bp fixed with the pre-cooled acetone for 10 min at room temperature. After the R: GCCGATCCACACGGAGTACT fixation, the slides were immersed in 3N HCL for 30 min at room temperature to expose the CpG sits. The slides were then treated with 1.0% hydrogen Abbreviations: F, forward; R, reverse.

Table 1 Clinical characteristics of the women enrolled in this study

Characteristics Normal pregnancy (n¼24) Early-onset preeclampsia (n¼10) Late-onset preeclampsia (n¼14)

Maternal age (years) 30.5652±4.0768 31.2000±5.1381 30.3571±3.6712 BMI (kg m–2) 24.5114±2.4750 24.1371±3.2172 23.5179±3.0814 SBP (mm Hg) 110.2174±8.3228 161.8000±29.0624* 158.2143±24.8540* DBP (mm Hg) 71.3043±5.6844 104.2000±15.9081* 106.0714±22.9698* 24 h proteinuria (g per 24 h) ND 3.9350±3.6700* 3.1658±3.1795* 50 g GCT (mmol lÀ1)7.4633±1.8116 7.2333±1.3614 8.1900±2.2650 Gestational age at delivery (day) 268.1739±12.8511 226.3000±8.7439* 257.9286±14.5996*

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; ND, not determined; SBP, systolic blood pressure; 50 g GCT, 50 g glucose challengetest. Data are expressed as means±s.d. and compared by the one-way analysis of variance or independent sample t-test. *Po0.05 vs. normal pregnancy.

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Table 3 Primers and PCR conditions for pyrosequencing analyses

Pyrosequencing Sequence Gene/Repeat PCR primers (5 ¢–3 ¢) primers (5 ¢–3 ¢) analyzed (5 ¢–3 ¢) PCR conditions PCR products

Alu F: biotin-TTTTTATTAAAAATATAAAAATT AATAACTAAAATTA G/AC/TG/AC/TG/ 95 1C for 90 s, 43 1Cfor60s, 144 bp R: CCCAAACTAAAATACAATAA CAAAC ACCACCA 72 1C for 120 s. (40 cycles)

LINE-1 F: TTTTTTGAGTTAGGTGTGGG GGGTGGGAGTGAT C/TGATTTTTTAGGTGC/ 95 1C for 30 s, 50 1C for 30 s, 249 bp R: biotin-TCTCACTAAAAAATACCAAACAA TGTTC/TG 72 1C for 30 s. (35 cycles)

Abbreviations: F, forward; R, reverse.

input RNA template, all results were normalized by using a geometric mean cycles of 95 1Cfor5s,601C for 35 s. HRM was carried out at 95 1C for 15 s, of the expression level of two internal control genes, GAPDH and b-actin.23 50 1C for 1 min and with a ramping from 60 to 95 1Crisingby0.11C every To compare the expression levels among different samples, the relative second as recommended by the manufacturer. HRM data were analyzed using mRNA level was calculated using the 2ÀDCT method recommended by the HRM software v2.0 (Applied Biosystems). The melting curves were Schmittgen et al.24 In our experiments, 2ÀDCT means 2À(CTDNMTÀCTcontrol) normalized by the calculation of two normalization regions before and after or 2À(CTH19ÀCTcontrol). In addition, product purity was confirmed by dissocia- the major fluorescence decrease representing the melting of the PCR product. tion curve analysis and agarose gel electrophoresis. This algorithm allows the direct comparison of the samples that have different starting fluorescence levels. Normalization regions were in the range 74–76 and Genomic DNA extraction and bisulfite treatment 85–88 1C. A differential profile was then evaluated for each sample by Genomic DNA was extracted from cells or frozen tissues using a genomic DNA comparing fluorescence at the melting point against the value of fluorescence extraction kit (ZR Genomic DNA II Kit; Zymo Research, Orange, CA, USA) of the negative control (unmethylated DNA). Values of relative fluorescent according to the manufacturer’s instructions. Bisulfite modification of genomic density for reference DNA standards were plotted against the corresponding DNA was carried out by using the EZ DNA Methylation-Gold Kit (Zymo methylation level to generate a typical standard curve, against which the Research) following the manufacturer’s instructions. methylation level of each unknown sample was evaluated.

Bisulfite PCR and pyrosequencing Statistical analysis ± DNA methylation level was quantified using the methods of bisulfite PCR and All data are presented as mean values s. d. calculated from three independent pyrosequencing.25,26 In brief, the bisulfite-modified DNA was PCR amplified experiments. Statistical comparison between groups was estimated using using the primers shown in Table 3. The PCR products were purified and two-sided Student’s t-test or one-way analysis of variance, and Po0.05 was quantified using the PSQ HS 96 Pyrosequencing System (Pyrosequencing, considered statistically significant. All statistical analyses were performed using Westborough, MA, USA). A biotin-labeled primer was used to purify the final the SPSS software for windows 13.0 (SPSS, Chicago, IL, USA) or GraphPad PCR product using sepharose beads. The PCR product was bound to Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). Streptavidin Sepharose High Performance (GE Healthcare, Milwaukee, WI, USA), and the Sepharose beads containing the immobilized PCR product were RESULTS purified, washed, denatured using a 0.2 mol lÀ1 NaOH solution, and washed Clinical characteristics of the subjects again using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing) according Of the 48 pregnant women enrolled in this study, 10 suffered from to the manufacturer’s recommendations. Subsequently, 0.3 mM pyrosequencing early-onset PE, 14 suffered from late-onset PE and 24 were normal primer was annealed to the purified single-stranded PCR product and pregnant subjects. The clinical characteristics of these study partici- pyrosequencing was done using the PSQ HS96 Pyrosequencing System. pants are described in Table 1. The systolic and diastolic blood Quantification of cytosine methylation was performed using the PSQ HS96A pressures as well as the 24 h urine protein content were significantly 1.2 software (Pyrosequencing). The ratio of C to T nucleotides was evaluated higher in the preeclamptic groups than in the control group. There for LINE-1 methylation, and ratio of G to A nucleotides was evaluated for Alu were no significant differences in age, body mass index and glucose methylation. The unmethylated human control DNA (EpiTect Control DNA; Qiagen, Hilden, Germany) was used to verify bisulfite conversion. tolerance (indicated by 50 g glucose challenge test) between the preeclamptic and the normal pregnant women. Methylation-sensitive high resolution melting analysis (MS-HRM) Global DNA methylation status in preeclamptic and normal MS-HRM was performed as previously described.27,28 Universal methylated and unmethylated genomic DNAs were purchased from Qiagen (EpiTect control placentas Control DNA; Qiagen), and mixed to create a serial of reference DNA standards 5-MC immunohistochemistry. Global DNA methylation was first containing methylation levels of 100, 75, 50, 25 and 0%. visually examined in frozen sections of placenta using an antibody PCR amplification and MS-HRM were performed on the Applied Biosys- directed against 5-MC. Figure 1 showed a representative pattern of tems 7500 real-time PCR system (Applied Biosystems). Primers for amplifying immunohystochemical staining for 5-MC in placenta villus from 5¢ promoter region of the H19 gene were described previously.29 The target preeclamptic and normal control women. HSCORE was used to region comprised the region from 9394 to 9529 within the sequence AF125183, semi-quantitatively evaluate the staining intensity. The HSCORE spanning the upstream of the H19 transcription start site and the downstream value of normal control, early-onset and late-onset preeclamptic of the CTCF7 zinc-finger binding site. PCR was performed using Fast EvaGreen placenta villus were 2.653±0.03697, 2.953±0.09952 and 2.763± Master Mix for quantitative PCR and HRM (Biotium, Hayward, CA, USA) in a 0.05038, respectively. Statistical analysis revealed that the intensity 20 ml reaction volume containing 10 ml2Â Fast EvaGreen Master Mix, 0.25 mM primers and 5 ng bisulfite-modified DNA. The primer sequences to bisulfite- was significantly higher in the early-onset preeclamptic placenta villus modified DNA were as follows: H19F, 5¢-GGGAGAGTTTGTGAGGT-3¢ and (P¼0.0036) as compared with the normal controls. The late-onset H19R, 5¢-AAATCCCCACAACCGCTAAAC-3¢. The 137-bp fragment was preeclamptic specimens exhibited relatively higher, but not statistically amplified from bisulfite-modified DNA. Cycling was at 95 1Cfor5min,40 significant, HSCORE values than the normal controls (P¼0.0826).

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Figure 1 Immunohistochemical staining for 5-methylcytosine in preeclamptic and control placentas. Figures show a relatively higher staining intensity in the early-onset preeclamptic placenta villus than in the normal controls. Bar¼100 mm.

Alu and LINE-1 pyrosequencing. To reliably compare the global methylation level in preeclamptic and normal placenta samples, we applied the pyrosequencing method to determine the methylation density in the Alu and LINE-1 repeats, which are surrogate markers of global methylation. In normal control, early-onset and late-onset preeclamptic placentas, Alu methylation levels were 0.2553±0.0091, 0.2952±0.0065 and 0.2786±0.0081, respectively, and LINE-1 methylation levels were 0.3067±0.0063, 0.3484±0.0084 and 0.3217 ±0.010, respectively. Statistical analysis showed that methylation of both Alu and LINE-1 repeats were signiﬁcantly higher in the early- onset preeclamptic placentas compared with the normal controls (P¼0.0054 for Alu; P¼0.0016 for LINE-1). Alu and LINE-1 methylation in the late-onset preeclamptic placentas were also relatively higher than the normal controls, but the changes did not reach statistical signiﬁcance (P¼0.1006 for Alu; P¼0.2136 for LINE-1) (Figure 2).

DNMTs mRNA expression. It is well established that DNA methylation at the CpG dinucleotides is catalyzed by various DNMTs, among Figure 2 Quantitation of DNA methylation using Alu (a)andLINE-1 (b) which DNA methyltransferase 1 (DNMT1), 3a and 3b are all highly pyrosequencing. Statistical analysis showed that methylation of both Alu and 30 expressed in human placenta. We then examined expression of LINE-1 repeats was significantly higher in the early-onset preeclamptic DNMTs mRNA in preeclamptic and control placentas using real- placentas as compared with normal controls (P¼0.0054 for Alu; P¼0.0016 time PCR (Figures 3a–c). After normalization by the control genes, for LINE-1). Alu and LINE-1 methylation in the late-onset preeclamptic GAPDH and b-actin, the expression of DNMT1 was significantly placentas were relatively higher than the normal controls, but the changes higher in the early onset (0.7523±0.2135), but not in the late-onset were not statistically significant (P¼0.1006 for Alu; P¼0.2136 for LINE-1). Box plots were used to show the methylation level. The line within each preeclamptic placentas (0.3344±0.1210) when compared with the box denotes the median. The crisscross denotes the mean. Limits of the ± normal controls (0.3020 0.0711). Placental DNMT3a and DNMT3b box denote the 25th and 75th percentiles. Whiskers denote the 5th and expressions did not show any statistically significant difference among 95th percentiles. *Po0.05 vs. Control. the three groups, though the level of DNMT3a in the early-onset ± preeclamptic placentas (0.0394 0.0093) was relatively higher than in H19 mRNA expression in the placentas. We further measured the ± normal controls (0.0201 0.0053). expression of H19 mRNA in preeclamptic and control placentas by Methylation status of the paternally imprinted H19 gene in using real-time PCR (Figure 4b). After normalization by the control preeclamptic and normal control placentas genes, GAPDH and b-actin,theH19 mRNA expression in control, ± The paternally imprinted H19 gene has been indicated to be critical to early-onset and late-onset preeclamptic placentas were 14.22 2.644, ± ± the early development of placenta. We further examined H19 mRNA 2.720 1.811 and 6.916 4.266, respectively. As expected, H19 mRNA expression and the methylation status in the promoter region of H19 level was appreciably lower in the early-onset preeclamptic placentas gene in preeclamptic placentas. than in controls (P¼0.0034). Though the level in the late-onset preeclamptic placentas was also lower than that in the normal MS-HRM for H19. We applied a rapid and sensitive tool, MS-HRM, controls, the change was not statistically significant (P¼0.1408). to selectively and reliably measure the degree of H19 methylation in placentas. The melt curve and standard curve of the method was DISCUSSION shown as Supplementary Figure 1. As shown in Figure 4a, methylation Epigenetic regulation in preeclamptic placenta has been scarcely levels in the promoter region of the H19 gene in normal control, early- investigated, and little evidence has been so far reported on the degree onset and late-onset preeclamptic placentas were 47.87±1.678, of global DNA methylation in this complex disease. In the present 57.19±2.068 and 51.51±2.182, respectively. Statistical analysis study, we measured the global DNA methylation level in preeclamptic revealed a statistically significant difference of H19 gene methylation placenta using immunohistochemistry for 5-MC and pyrosequencing in the early-onset preeclamptic placentas as compared with the normal of Alu and LINE-1 repeats, which are two surrogate markers of global controls (P¼0.0023). methylation. The data consistently revealed a marked hypermethyla-

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Figure 3 DNMT1 (a), DNMT3a (b)andDNMT3b (c) mRNA levels in placentas were determined by real-time PCR. The expression of DNMTs was normalized by the control genes GAPDH and b-actin. The expression of DNMT1 was signiﬁcantly higher in the early onset, but not in the late-onset preeclamptic placentas when compared with the normal controls. Placental DNMT3a and DNMT3b expressions did not show any statistically signiﬁcant difference among the three groups, though the level of DNMT3a in the early-onset preeclamptic placentas was relatively higher than in normal control. *Po0.05 vs. Control.

methylation patterns during cell division in mammalian cells. On the other hand, DNMT3a and 3b are specific methyltransfersase involved in establishing DNA methylation patterns during embryonic development.31 Our results thus indicated that the significantly higher expression of DNMT1 may substantially contribute to the global hypermethylation in the early-onset preeclamptic placenta. Recent studies may suggest cues as to the factors that may cause high DNMT1 expression and global hypermethylation in PE patients. Folate, in the form of 5-methyltetrahydrofolate, is involved in remethylation of homocysteine to methionine, which is a precursor of SAM, the primary methyl group donor for DNA methylation. Adult rats subject to a short-term folate-deficient diet exhibited upregulation of DNMT as well as genomic DNA hypermethylation in the liver.32 Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine.33 Feeding pregnant rats with a choline-deficient diet led to increased global DNA methylation in fetal liver and brain.34 Deficiency of methyl group donors like folate or choline could result in hypomethylation of the regulatory CpGs within the DNMT1 gene, leading to its overexpression and subsequent increased global Figure 4 H19 methylation and mRNA expression levels in control and DNA methylation.35 Furthermore, Ray et al. reported that pregnant preeclamptic placentas. (a) H19 methylation levels were evaluated by women with low levels of circulating folate had an increased risk of methylation-sensitive high resolution melting (MS-HRM). The degree of developing PE.36 Lastly, results from two independent laboratories methylation of clinical samples was evaluated by comparison with the demonstrated that women with homozygous mutation of the T677 standard curve. Statistical analysis revealed increased methylation of the H19 gene in the early-onset preeclamptic placentas when compared with allele in the 5, 10-methylenetetrahydrofolate reductase (MTHFR)gene, the normal controls (P¼0.0023). The late-onset preeclamptic placentas also an important enzyme in folate metabolism, had a significantly showed hypermethylation of the H19 gene when compared with the normal increased risk of PE.37,38 Therefore, it may be worth investigating controls, but the change was not statistically significant (P¼0.1944). (b) whether abnormality of folate or choline metabolisms is involved in The expression of H19 was measured by real time PCR, and normalized by the onset of PE via interfering with the status of DNA methylation the control genes GAPDH and b-actin. H19 mRNA level was appreciably during placentation. lower in the early-onset preeclamptic placentas than in controls (P¼0.0034). Though the level in the late-onset preeclamptic placentas was Appropriate DNA methylation is indispensable for normal placenta 39 also lower than that in the normal controls, the change was not statistically development and function. Anumberofstudieshavesuggested significant (P¼0.1408). *Po0.05 vs. Control. critical links between alterations of appropriate epigenetic regulation in the placenta and diseases of gestation and early life. General disruption of DNA methylation consequent to certain clinical drug tion in the genome of the early-onset preeclamptic placentas as treatments has been shown to inhibit invasiveness of trophoblast cells compared with control placentas. It is well established that DNA and therefore to disrupt placental development.40,41 Specific genetic methylation at the CpG dinucleotides is catalyzed by various DNMTs, approaches have shown that lack of DNMT1 or DNMT3L genes among which DNMT1, 3a and 3b are all highly expressed in human results in impaired trophoblast differentiation and aberrant placenta placenta.30 DNMT1 is usually regarded as a maintenance methyl- development.42,43 A recent report has revealed that LINE-1 sequences transferase being responsible for accurately replicating genomic DNA are significantly hypermethylated in partial hydatidiform moles tissues

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relative to normal placentas.44 Partial hydatidiform moles is charac- experiments, we examined the expression of DNMTs and H19 as well terized by placenta overgrowth, trophoblastic hyperplasia and as global methylation and H19 methylation in placentas derived from abnormal or absent fetus.45 These placenta phenotypes are to some six pre-term birth patients who delivered at gestational weeks 32–35. extent similar to the observations in preeclamptic placenta where No evident differences in either DNMTs and H19 expression or global immature intermediate trophoblasts are over proliferating.46 Cases of methylation and H19 methylation were found between these samples PE in combination with partial hydatidiform mole have also been and term placentas (Supplementary Figure 2). Therefore, we used reported in literatures.47,48 Taken together, these data demonstrate an normal term placentas as universal controls in our study. Meanwhile, epigenetic disorder in placentas of women who suffered from gesta- the extensive microarray data of Winn et al. did not show the tional diseases such as PE and partial hydatidiform mole. It remains to expression change of DNMTs and H19 genes during various develop- be investigated whether global DNA hypermethylation in placenta is a mental stages in human placentas.54 What’s more, Fuke et al. demon- causal factor for gestational diseases. strated that the global methylation (as measured by 5-MC content) in The paternally imprinted H19 gene is transcribed as an untranslated human placenta increased on average by 10% at the first half of RNA that may serve as a riboregulator. H19 is located in close pregnancy, but exhibited slight gestation-dependent difference at proximity to the maternally imprinted IGF-2 gene on chromosome weeks 32 to term.55 The early-onset, late-onset preeclamptic placentas 11p15.517 and the H19 null mice display hyperplasia of all layers of the and normal control ones used in this study were at average gestational placenta.18 These data strongly indicate that the H19 gene is critical to weeks 32.2, 36.8 and 38.3, respectively. Taken these evidences together, the early development of placenta. The actual functions of the H19 we propose that the difference in the duration of the pregnancy gene are still unclear. During pregnancy, the H19 gene in the devel- according to the disease status may have little influence on both global oping placenta can be expressed either biallelically or monoallelically. methylation and H19 methylation and expression. Its biallelic expression is confined to the placenta until 10 weeks of In general, the present study is the first report on the change of gestation, after which it is predominantly, but not exclusively, global DNA methylation and H19 gene methylation in human expressed from the maternal allele, and its paternal allele is proved preeclamptic placenta. The influence of such epigenetic change on to be methylated.49 Some studies suggested that H19 might act as an placenta development as well as the occurrence of gestational diseases oncogene in human gestational trophoblastic tumors, based on the needs further investigations. evidences that high level and biallelic expression of H19 gene was observed in human choriocarcinoma cells, as well as that H19 gene CONFLICT OF INTEREST expression was much higher in the tumors formed by choriocarci- The authors declare no conflict of interest. noma cells injected in nude mice.50 In human placenta, H19 expression was most pronounced in the invasive intermediate trophoblasts and villous cytotrophoblasts, whereas its expression was negligible in ACKNOWLEDGEMENTS syncytiotrophoblasts. Such a differential expression pattern of H19 The work was supported by the grants from the Chinese National Special Fund may be consistent with its proposed role in regulating the invasive for Basic Research Project (No: 2011CB944400), National Natural Sciences property of trophoblasts.19,20 This possibility is supported by the data Foundation (No: 30530760) and the Knowledge Innovation Program in from Walsh et al., who showed an invasion-promoting effect of H19 Chinese Academy of Sciences (No: KSCX2-EW-R-06). gene in trophoblastic cells.51 The trophoblasts in preeclamptic placenta showed trophoblastic hyperplasia, over-proliferated immature intermediate trophoblasts and impaired trophoblast invasion.46 The hyper- methylationandreducedexpressionoftheH19 gene in early-onset 1 Roberts JM, Cooper DW. Pathogenesis and genetics of pre-eclampsia. Lancet 2001; 357:53–56. preeclamptic placentas as reported in the present study, may indicate a 2 Sibai BM, Ewell M, Levine RJ, Klebanoff MA, Esterlitz J, Catalano PM, Goldenberg RL, role of H19 in the abnormal proliferation and invasion phenotypes of Joffe G. Risk factors associated with preeclampsia in healthy nulliparous women. The Calcium for Preeclampsia Prevention (CPEP) Study Group. Am J Obstet Gynecol 1997; the preeclamptic trophoblasts. Indeed, recent data suggest that H19 177: 1003–1010. might regulate expressions of specific target genes by acting as the 3 Roberts JM. Endothelial dysfunction in preeclampsia. Semin Reprod Endocrinol 1998; precursor of microRNA-675.52 To better address this issue, we are 16:5–15. 4 Hladunewich M, Karumanchi SA, Lafayette R. Pathophysiology of the clinical currently performing further studies on the molecular mechanisms manifestations of preeclampsia. Clin J Am Soc Nephrol 2007; 2: 543–549. underlying H19 gene functions in human placental trophoblasts. 5 Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science It has been suggested that early-onset and late-onset PE may have 2005; 308: 1592–1594. 6 Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, different etiologies and clinical expression, and should be regarded as Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental different forms of the disease.53 The different methylation status in soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. JClinInvest2003; 111: 649–658. placental genome as well as in the H19 gene as observed in this study is 7 Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, Bdolah Y, Lim KH, likely supporting this idea. However, for the late-onset preeclamptic Yuan HT, Libermann TA, Stillman IE, Roberts D, D¢Amore PA, Epstein FH, Sellke FW, placentas, both the global methylation and H19 gene methylation Romero R, Sukhatme VP, Letarte M, Karumanchi SA. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med 2006; 12:642–649. levels are in between the levels observed in the normal gestations and 8 Serman L, Vlahovic M, Sijan M, Bulic-Jakus F, Serman A, Sincic N, Matijevic R, Juric- early-onset PE. Therefore, it is quite possible that early-onset and late- Lekic G, Katusic A. The impact of 5-azacytidine on placental weight, glycoprotein onset PE represent two degrees of severity of the disease, and maybe a pattern and proliferating cell nuclear antigen expression in rat placenta. Placenta 2007; 28: 803–811. pathological continuum. Patients with early-onset PE may have severe 9 Ohgane J, Wakayama T, Senda S, Yamazaki Y, Inoue K, Ogura A, Marh J, Tanaka S, genetic or epigenetic disorders such as aberrant methylation in both Yanagimachi R, Shiota K. The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice. Genes Cells 2004; 9: genome and imprinted H19 gene, which can disturb the proper early 253–260. development of placenta during pregnancy. 10 Chim SS, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, Oudejans CB, Ding C, Lo YM. One problem in our study is the difference in the duration of the Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci USA 2005; 102: 14753–14758. pregnancy according to the disease status, which may cause gestation- 11 Chelbi ST, Mondon F, Jammes H, Buffat C, Mignot TM, Tost J, Busato F, Gut I, dependent variation in DNA methylation levels. In our preliminary Rebourcet R, Laissue P, Tsatsaris V, Goffinet F, Rigourd V, Carbonne B, Ferre F, Vaiman D.

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